In view of the wide distribution or glycoproteins and the important biological role which is being attributed to their saccharide units, it is important to elucidate the enzymatic steps involved in their synthesis. Indeed, the attachment of carbohydrate and its subsequent processing is one of the major post translational modifications which a protein may undergo on its way to becoming a biol gically relevant molecule and one which may be susceptible to regulation as well as to alteration in disease. The studies proposed in this renewal application aim to identify and characterize enzymes, intermediates, subcellular organelles and regulatory factors involved in glycoprotein assembly. For this purpose tissues (e.g., thyroid, kidney) of higher animals active in the biosynthesis of defined secretory and membrane glycoproteins will be used at several levels of organization. The metabolism and role of the recently described glucose-containing dolichol pyrophosphate-linked oligosaccharide intermediates will be studied in detail including their assembly by glycosyltransferases, their attachment to protein and their subsequent processing and modification to yield the two major types of asparagine-linked carbohydrate units. The transfer to protein will be studied in cell-free translation-glycosylation systems with various mRNAs. Individual processing enzymes (glucosidases, mannosidases) will be characterized and their concerted action investigated by pulse-chase studies in thyroid slices and free cells. Hormonal and nutritional influences on these steps of carbohydrate unit assembly and processing as well as on the synthesis of the dolichol carrier will be studied in whole animals and in vitro. A cell-free translation system (wheat germ) will also be employed to identify the unmodified polypeptide precursor(s) of the collagen-like glycoproteins of basement membranes, which in turn will be used for subsequent hydroxylation and glycosylation reactions. The effect of the diabetic state on renal basement membrane synthesis will be assessed from the level of specific mRNA available for translation in such a cell-free system.